Thrombotic thrombocytopenic purpura (TTP) is a catastrophic and potentially fatal microvascular thrombotic disorder associated with congenital or acquired deficiency of the plasma metalloprotease ADAMTS-13. The only known function of ADAMTS-13 is to cleave von Willebrand factor (VWF), a multimeric protein that when newly secreted spontaneously binds and activates platelets, accounting for the systemic thrombosis. The newly secreted VWF is large and tremendously adhesive, and has been named unusually large or ultra-large VWF (ULVWF). ADAMTS-13 converts ULVWF to smaller and less adhesive forms that circulate in the blood. Although ADAMTS-13 deficiency is necessary for the clinical manifestation of TTP, it is not sufficient, as is clear from the fact that patients congenitally deficient in the enzyme often do not manifest TTP until the second or third decade of life. We have shown that ULVWF exists in a different conformation than plasma VWF, a conformation that allows it to spontaneously bind the platelet receptor glycoprotein (GP) Ib1. We hypothesize that this alternative conformation is based on a different pattern of disulfide bonds in the two forms of VWF and that the hyperreactive conformation can be converted to the less reactive conformation not only by proteolysis but also by disulfide isomerization or reduction. The experiments proposed in this application will test that hypothesis and will evaluate a potential therapy for TTP based on that hypothesis. We have three Specific Aims: 1) To determine the molecular nature of the hyperreactive form of VWF by determining the disulfide-bonded structure of the ULVWF A1-A2-A3 region. We will compare the arrangement of disulfide bonds in plasma VWF and ULVWF within the A1-A2-A3 region, which contains binding sites for GP Ib1 and collagen, as well as the ADAMTS-13 cleavage site. We expect that this will shed light on the basis of ULVWF hyperreactivity. 2) To examine the role of VWF disulfide isomerization/reduction in VWF reactivity. Here, we will examine how different redox conditions and disulfide reductases/isomerases will affect the functions of VWF and ULVWF. 3) To evaluate further the effect of N-acetylcysteine as a potential treatment for thrombotic thrombocytopenic purpura. We have evidence that N-acetylcysteine can reduce the size of ULVWF multimers and modulate the functions of both plasma VWF and ULVWF. We will explore the molecular basis of this effect and examine whether this agent can prevent or treat induced TTP in a mouse model of ADAMTS-13 deficiency. PUBLIC HEALTH RELEVANCE: In this application, we endeavor to find out why a plasma protein, von Willebrand factor (VWF), is very sticky for platelets when it is first released from the cells in which it is made. If this protein is not normally processed, it causes a severe blood clotting disorder that often leads to death of the affected patient. The studies we propose will help us learn how the new VWF is different from that VWF that has been processes and we will learn how it is processed. In this way, we will be able to understand and treat disorders of blood clotting.